Previous work in this laboratory has shown that during bacterial growth conditions under which glutamine synthetase activity is being lost, antigencially cross-reacting material initially persists, suggesting that inactivation precedes degradation. Examination of the inactivating activity of cell-free extracts led to the development of a model inactivating system including only ascorbic acid, iron, and oxygen as necessary components. Amino acid analysis revealed the loss of one histidine residue per subunit in the inactivated species. Further analysis was attempted exploiting the ability of the protease subtilisin to clip the glutamine synthetase subunit in two. It was discovered that the inactivated glutamine synthetase was more susceptible to proteolysis than the native enzyme, both in the presence and absence of substrate (ammonium sulfate). It was shown in the case of the proteolysis of the native enzyme that the loss of intact subunit precisely paralleled the attendant loss of enzymatic activity, suggesting that the cleavage of the susceptible peptide bond in this protein could be followed by enzymatic assay. In so doing, it was determined that in this interaction, the Km of subtilisin for glutamine synthetase subunit is about 0.8 Mumolar.